CN107400666A

CN107400666A - A kind of aminopeptidase and its encoding gene and application

Info

Publication number: CN107400666A
Application number: CN201710812644.4A
Authority: CN
Inventors: 叶茂; 邓毛程; 李静; 张远平; 陈维新; 刘慧平
Original assignee: Guangdong Industry Technical College
Current assignee: Guangdong Industry Technical College
Priority date: 2017-09-11
Filing date: 2017-09-11
Publication date: 2017-11-28
Anticipated expiration: 2037-09-11
Also published as: CN107400666B

Abstract

The invention discloses a kind of aminopeptidase and its encoding gene and application.The amino acid sequence of the aminopeptidase is as shown in SEQ ID NO.1.Present invention also offers the nucleotide sequence for encoding the aminopeptidase.The aminopeptidase has the function that almost all of amino acid residue can be hydrolyzed have good application potential, and all significant to abundant aminopeptidase family member, exploitation China's traditional condiment environmental resource.Therefore, it can use it for preparing flavor peptide, flavor peptide can be made by the steps：Aminopeptidase provided by the invention is added in the hydrolysis reaction system containing protein substrate, 2~6h of enzymatic hydrolysis reaction is carried out under the conditions of 25~35 DEG C, obtains corresponding flavor peptide.

Description

A kind of aminopeptidase and its encoding gene and application

Technical field

The invention belongs to genetic engineering and enzyme engineering field, more particularly to a kind of aminopeptidase and its encoding gene and application.

Background technology

Aminopeptidase is a kind of N-terminal sequential hydrolysis amino acid from polypeptide chain, makes the hydrolase of amino acid separate out one by one, The general more wide spectrum of its substrate-function scope, can not only hydrolyzed peptide, and complete protein molecule can be hydrolyzed.Aminopeptidase It is mainly used in protein hydrolysis, prepared by flavor peptides and biologically active polypeptide and the field such as biology and medical science：Work as protein When macromolecular digests, the hydrophobic amino acid contained in peptide chain is exposed, and contacts taste bud and bitter taste is presented, and these hydrophobicitys Amino acid is normally at peptide chain end, and utilizes the further aminosal hydrolyzate of aminopeptidase, can remove its bitter taste；With egg White enzyme is used in combination, and in the preparation process of soy sauce brewing, fish sauce production and other proteolysis products, can not only improve egg The utilization rate of white matter, and the delicious compounded amino acid hydrolysis liquid of unique flavor, flavour can be formed；Utilize the enzymolysis system of aminopeptidase The active peptide of the characteristics such as standby anticancer, anti-oxidant, antibacterial；In terms of medical usage, leucine aminopeptidase etc. also acts as protein The molecular tool of sequencing, prolidase can effectively remove neurotoxicity gas and organophosphorus pesticide, can not only be used for curing Antidote on, environment disinfectant can be used as again.Thus, aminopeptidase has very high researching value and broader practice Prospect.

Metagenomics (Metagenomics) are the fast developments recently as microbiology and modern life science The emerging science and technology risen.Its basic research thinking is the genomic DNA of all microorganisms in direct extraction environment, Suitable carrier is cloned into, builds grand genomic library, the information nucleic acid of whole microorganisms in environment is collected together, and transports Useful enzyme, antibiotic, active material etc. are obtained from library with sequence screening or functional screening mode.It is this completely independent of The separation of microorganism and technique extension microbiological Research Thinking and the method for culture in environment, it is to be given birth to naturally from different New genetic resources is found in Anticipated transient without scram in border and active material provides strong technological means.

Up to the present, the research of new aminopeptidase gene is found from grand genomic library, there is no the report of correlation both at home and abroad Road.

The content of the invention

The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, there is provided a kind of aminopeptidase.The enzyme is to N- Terminal amino acid residue selectivity is low, can hydrolyze almost all of amino acid residue, to abundant aminopeptidase family member, develops new The approach of proteolysis into flavor peptides is all significant.

Another object of the present invention is to provide to encode the gene of the aminopeptidase.

It is still another object of the present invention to provide the application of the aminopeptidase.

The purpose of the present invention is achieved through the following technical solutions：A kind of aminopeptidase, its amino acid sequence are as follows：

MNFQVQDAAIVIEAVIVALAFFAEKIIFAQQLDAAITGSLQALLAHKGISTVGYAVSLLHSLAGLNVDRYYVGLEGQ QLAYGIQTLLRKAGDAFTTIKGGAVSDEQIKLPSFTKQRLDAAVLHIAVVSPGHGAYELTYSKKKDKQELFEFTAIA LDDAQLDLEGFISVGDVTGVLLNFARQLGNAHPHVLTPAIKTAEEAVALFGKTDYDVKLDKEQMEGIGNGSLLAVKK GSENIPRLIVLTYNGKEDAAPVVALVGPGITFDSGGYSLKTRQGMEFMKFDMGCGAAVGGTVEAIGHLLPPKNIVGV IPASDNIVNGEAISPLDVGTSLSGYTIEVMNTFPEGRLLLADAVFYAYQDGPGVLADVATLTGAYIVVLGAHKTAGA MINPGILFNQLLDASEEVDEPAGLPAIWGTEGGVVTSDVADLPNYGGRDGTALFEGMFLTEEPENTPWLYLDIAGGS RIEDAVLIGPDGAEGLLVPTLVDLLTREGEAEA。

A kind of nucleotide sequence, it is the nucleotide sequence of the described aminopeptidase of coding；Preferably intronless, have The nucleotide sequence of the complete opening code-reading frames of 1485bp；Sequence more preferably as follows：

ATGAACTTCCAGGTTCAGGACGCTGCTATCGTTATCGAAGCTGTTATCGTTGCTCTGGCTTTCTTCGCTGAAAAAAT CATCTTCGCTCAGCAGCTGGACGCTGCTATCACCGGTTCTCTGCAGGCTCTGCTGGCTCACAAAGGTATCTCTACCG TTGGTTACGCTGTTTCTCTGCTGCACTCTCTGGCTGGTCTGAACGTTGACCGTTACTACGTTGGTCTGGAAGGTCAG CAGCTGGCTTACGGTATCCAGACCCTGCTGCGTAAAGCTGGTGACGCTTTCACCACCATCAAAGGTGGTGCTGTTTC TGACGAACAGATCAAACTGCCGTCTTTCACCAAACAGCGTCTGGACGCTGCTGTTCTGCACATCGCTGTTGTTTCTC CGGGTCACGGTGCTTACGAACTGACCTACTCTAAAAAAAAAGACAAACAGGAACTGTTCGAATTCACCGCTATCGCT CTGGACGACGCTCAGCTGGACCTGGAAGGTTTCATCTCTGTTGGTGACGTTACCGGTGTTCTGCTGAACTTCGCTCG TCAGCTGGGTAACGCTCACCCGCACGTTCTGACCCCGGCTATCAAAACCGCTGAAGAAGCTGTTGCTCTGTTCGGTA AAACCGACTACGACGTTAAACTGGACAAAGAACAGATGGAAGGTATCGGTAACGGTTCTCTGCTGGCTGTTAAAAAA GGTTCTGAAAACATCCCGCGTCTGATCGTTCTGACCTACAACGGTAAAGAAGACGCTGCTCCGGTTGTTGCTCTGGT TGGTCCGGGTATCACCTTCGACTCTGGTGGTTACTCTCTGAAAACCCGTCAGGGTATGGAATTCATGAAATTCGACA TGGGTTGCGGTGCTGCTGTTGGTGGTACCGTTGAAGCTATCGGTCACCTGCTGCCGCCGAAAAACATCGTTGGTGTT ATCCCGGCTTCTGACAACATCGTTAACGGTGAAGCTATCTCTCCGCTGGACGTTGGTACCTCTCTGTCTGGTTACAC CATCGAAGTTATGAACACCTTCCCGGAAGGTCGTCTGCTGCTGGCTGACGCTGTTTTCTACGCTTACCAGGACGGTC CGGGTGTTCTGGCTGACGTTGCTACCCTGACCGGTGCTTACATCGTTGTTCTGGGTGCTCACAAAACCGCTGGTGCT ATGATCAACCCGGGTATCCTGTTCAACCAGCTGCTGGACGCTTCTGAAGAAGTTGACGAACCGGCTGGTCTGCCGGC TATCTGGGGTACCGAAGGTGGTGTTGTTACCTCTGACGTTGCTGACCTGCCGAACTACGGTGGTCGTGACGGTACCG CTCTGTTCGAAGGTATGTTCCTGACCGAAGAACCGGAAAACACCCCGTGGCTGTACCTGGACATCGCTGGTGGTTCT CGTATCGAAGACGCTGTTCTGATCGGTCCGGACGGTGCTGAAGGTCTGCTGGTTCCGACCCTGGTTGACCTGCTGAC CCGTGAAGGTGAAGCTGAAGCT。

Above-mentioned nucleotide sequence can be obtained by chemical synthesis process, can also be made by the steps to obtain：

First, traditional fermented food environmental sample is extracted, total genome is extracted and purifies；

2nd, total genome after purification is handled through Sau3AI digestions；

3rd, digestion products are connected on pUC18/BamHI (BAP) carrier, built in electroporated bacillus coli DH 5 alpha Traditional fermented food environment macro genomic library, ammonia peptide is screened from library with liquid selective medium and solid selection medium Enzyme positive is cloned, and positive clone molecule is obtained by high flux screening；

4th, design primer is compared through sequencing and Blast, using the plasmid of positive clone molecule as template, with designed primer Chain type enzymatic polymerization reaction is carried out, so as to clone to obtain above-mentioned nucleotide sequence.

Traditional fermented food described in step 1 includes soy sauce, fermented bean curd, beans sauce and sausage etc..

The composition of Selective agar medium described in step 3 is as follows：100 μ g/ml ampicillins, the bright ammonia of 10mmol/L L- Without amino acid yeast nitrogen, solvent is water by acid -4- nitroanilines, 1 (w/v) % glucose and 0.5 (w/v) %.

Designed primer described in step 4 is as follows：

Sense primer F1：5’-CGCGGATCCATGAACTTCCAGGTTCAG-3’；

Anti-sense primer F2：5’-CGGAAGCTTAGCTTCACCTTCACGGTCA-3’.

In addition, present invention also offers the preparation method of above-mentioned aminopeptidase, can be obtained by chemical synthesis process, or adopt Express to obtain with above-mentioned nucleotide sequence, comprise the following steps：

(1) nucleotide sequence is cloned into expression vector, then be transferred in expression cell, obtained containing the thin of recombinant vector Born of the same parents；

(2) cell containing recombinant vector obtained to step (1) is cultivated, and is induced through IPTG, separated from culture, Purifying, obtain aminopeptidase.The aminopeptidase has the function that almost all of amino acid residue can be hydrolyzed.

Expression vector described in step (1) is preferably pET-32a (+).

Expression cell described in step (1) is preferably e. coli bl21 (DE3).

The culture medium of culture described in step (2) is preferably the LB fluid nutrient mediums containing 100 μ g/ml ampicillins.

Induction described in step (2) comprises the following steps that：When the cell growth containing recombinant vector to OD₆₀₀=1.0 When add IPTG to final concentration 0.8mM, 20 DEG C, cultivate 20h under 200r/min.

The mode of separation described in step (2) is preferably to centrifuge.

In addition, the application present invention also offers described aminopeptidase in flavor peptide is prepared, preferably includes following steps： Described aminopeptidase is added in the hydrolysis reaction system containing protein substrate, it is anti-that enzyme hydrolysis is carried out under the conditions of 25~35 DEG C 2~6h is answered, obtains corresponding flavor peptide.

Described protein substrate includes soybean protein, zein, wheat gluten and Yeast protein etc..

The pH value of described hydrolysis reaction system is 6~8；Preferably 7.

Cushioning liquid in described hydrolysis reaction system is preferably phosphate buffer.

Mass concentration of the described protein substrate in described hydrolysis reaction system is 1%~10%；Preferably 1%.

The present invention is had the following advantages relative to prior art and effect：

(1) compared to prior art, the present invention applies technique of metagenome from China's traditional condiment environment macro genome The aminopeptidase that library obtains, it was found that the enzyme has the function that almost all of amino acid residue can be hydrolyzed have good answer With potentiality, and it is all significant to abundant aminopeptidase family member, exploitation China's traditional condiment environmental resource.

(2) originally return by studying optimum condition of the enzyme in protein hydrolysate generates flavor peptide, it was found that Hydrolysis using albumen such as soybean protein, zein, wheat gluten, Yeast proteins as substrate raw material is generated in flavor reactive polypeptide, The aminopeptidase of the present invention carries out enzymic catalytic reaction 2-6h under the conditions of 25-35 DEG C, and the mass concentration of the substrate is 1%~ 10%, generation flavor peptide, flavor peptide molecular weight≤3000Da, sense organ can efficiently, rapidly be hydrolyzed using above-mentioned reaction condition Identification has characteristic flavor on basis, has good application value.

Brief description of the drawings

Fig. 1 is the SDS-PAGE of aminopeptidase purifying provided by the invention；Wherein, swimming lane M is albumen Marker；Swimming Road 1 and 2 is respectively recombinant protein crude enzyme liquid (not purifying), and swimming lane 3 is albumen after purification.

Fig. 2 is the substrate specificity measurement result figure of the aminopeptidase of purifying.

Fig. 3 is the optimal reactive temperature measurement result figure of the aminopeptidase of purifying.

Fig. 4 is the optimal reaction pH value measurement result figure of the aminopeptidase of purifying.

Embodiment

With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.

The acquisition of the aminopeptidase gene of embodiment 1 and the checking of protein hydrolysate generation flavor peptide function

1st, the acquisition of aminopeptidase gene

From China's traditional fermented food --- sauce fermentation environmental sample (i.e. moromi), build fermented food environment macro gene Group library.With containing 100 μ g/ml ampicillins, 10mmol/L L-Leu -4- nitroanilines, 1 (w/v) % glucose and Culture mediums of 0.5 (w/v) % without amino acid yeast nitrogen (YNB) alternatively culture medium.Cell in library is connect with photocopy Kind pin is copied on solid selective medium flat board, is cultivated 3 days at 37 DEG C, and it is existing that screening periphery of bacterial colonies has yellow hydrolysis to iris out Positive clone molecule, aminopeptidase positive colony is screened from library.

The plasmid for extracting above-mentioned positive clone molecule is sent to sequencing company and is sequenced, and obtains the nucleic acid sequence of an aminopeptidase Row, the sequence are named as an_105 by 1485 base compositions, its nucleotide sequence as shown in SEQ ID NO.2；The nucleic acid The polypeptide of coding, containing 495 amino acid, its amino acid sequence is named as AN_105 as shown in SEQ ID NO.1.

2nd, the clone of genetic fragment

According to nucleic acid an_105 sequences, amplimer is designed：Sense primer F1：5’- CGCGGATCCATGAACTTCCAGGTTCAG-3 ' (underscore is BamHI restriction enzyme sites sequence)；Anti-sense primer F2：5’- CGGAAGCTTAGCTTCACCTTCACGGTCA-3 ' (underscore is HindIII restriction enzyme sites sequence).

Using the plasmid of positive clone molecule as template, using F1 and F2 as primer, enter performing PCR amplification, reaction system is： PrimeSTAR^TMThe μ l of HS DNA Ploymerase (2.5U/ μ l) 0.3,5 × buffer solution 6 μ l, dNTP Mix (2.5mM) 2.4 μ l, The μ l of template (100ng/ μ l) 0.6, primers F 1 and each 0.3 μ l of F2 (10mM), ultra-pure water complement to 50 μ l.PCR reaction conditions：95℃ Pre-degeneration 2min；98 DEG C of denaturation 10s, 68 DEG C of annealing 15s, 72 DEG C of extension 1.5min, 30 circulate；72 DEG C extension 10min, 4 DEG C.

By PCR primer with BamHI and HindIII double digestion 16h are used after PCR primer Purification Kit, with passing through together PET-32a (+) (Invitrogen) expression vector of sample processing is attached, electricity conversion to expressive host e. coli bl21 (DE3), by resistance screening, picking positive clone molecule, extract DNA, carry out sequence verification find its nucleotide sequence with Sequence in SEQ ID NO.2 is identical, can effectively obtain target gene fragment.

3rd, Aminopeptidase A N_105 acquisition is recombinated

The inoculation through sequence verification correct plasmid to the LB Liquid Cultures for containing 100 μ g/ml ampicillins will be contained In base, 30 DEG C, cultivate 14h under 250r/min, contain 100 μ g/ml ammonia benzyl moulds by what 2 (v/v) % inoculum concentration was forwarded to 100ml In the LB fluid nutrient mediums of element, when growing to OD₆₀₀Isopropylthio-β-D-galactoside is added when=1.0 to final concentration 0.8mM, 20 DEG C, 20h is cultivated under 200r/min, 12000r/min centrifugation 5min, supernatant is abandoned, thalline is resuspended in 20ml 50mM In Tri-HCl (pH7.5), crush thalline with sonicator and (200w, 10 seconds time, be spaced 15 seconds, whole 5 minutes, ice Bath), 4 DEG C, 13000r/min centrifugation 15min, supernatant is collected, that is, obtains restructuring aminopeptidase crude enzyme liquid.

The His label protein purification kits Proband of aminopeptidase crude enzyme liquid Invitrogen companies will be recombinated Purification system carry out purification of recombinant proteins, and concrete operation step is carried out by the said firm's product description.After purification Recombinant protein liquid nitrogen it is quick-frozen, preserve into ultra low temperature freezer.The concentration of recombinant protein after purification is 290 μ g/ml.

Crude enzyme liquid and albumen after purification are determined by polyacrylamide gel electrophoresis, as a result as shown in Figure 1.It is it can be seen that logical Cross His label protein purification kit Proband Purification system and can succeed and purify to obtain this hair from crude enzyme liquid Bright aminopeptidase.

4th, the checking of protein hydrolysate generation flavor peptide function

By the above-mentioned restructuring aminopeptidase crude enzyme liquid 2ml or μ l of the enzyme liquid of purifying 20,8ml pH7.0 phosphate buffers are added (0.2M biphosphate sodium water solution 39ml and 0.2M disodium hydrogen phosphate aqueous solution 61ml mixing) is used as reaction medium, tests respectively Different substrates (albumen such as soybean protein, zein, wheat gluten, Yeast protein), concentration are 1 (w/w) %, 30 DEG C of water-baths After reacting 6h, hydrolysis generates corresponding flavor peptide, flavor peptide molecular weight≤3000Da, and naked eyes evaluation has characteristic flavor on basis.Product leads to Cross GC-MS and carry out Structural Identification.

Embodiment 2 recombinates the analysis of Aminopeptidase A N_105 substrate specificities

100mM Tris-HCl buffer solutions (pH7.0), 1mM substrates, the μ l of enzyme liquid 10 of purifying are added, extinction is determined at 35 DEG C Value A_405nm.Determining substrate is：L-Leu -4- nitroanilines (L-Leu-PNA, L-Leucine p-nitroanilide Hydrochloride, CAS 16010-98-3), L-Methionine -4- nitroanilines (L-Met-PNA, L-Methionine p- Nitroanilide, CAS 6042-04-2), L-PROLINE -4- nitroanilines (L-Pro-PNA, L-Proline p- Nitroanilide trifluoroacetate salt, CAS 108321-19-3), 1B -4- nitroanilines (L- Lys-PNA, L-Lysine p-nitroanilide dihydrobromide, CAS40492-96-4), L-arginine -4- nitros Aniline (L-Arg-PNA, L-Arginine p-nitroanilide dihydrochloride, CAS 40127-11-5), N- penta Two acyls-L-phenylalanine -4- nitroanilines (L-Phe-PNA, N-Glutaryl-L-phenylalanine p- Nitroanilide, CAS 5800-34-0), Pidolidone -4- nitroanilines (L-Glu-PNA, L-Glutamic acid γ - (p-nitroanilide) hydrochloride, CAS67953-08-6), D-phenylalanine-valine -4- nitroanilines (D- Phe-Val-PNA, D-Phe-Val-p-nitroanilide, CAS 108321-89-7), D-Val-leucine-lysine- 4- nitroanilines (D-Val-Leu-Lys-4-PNA, D-Val-Leu-Lys p-nitroanilide dihydrochloride), N- benzoyls-tyrosine -4- nitroanilines (N-Benzoyl-L-Tyr-PNA, N-Benzoyl-L-tyrosine p- nitroanilide,CAS 6154-45-6).As a result as shown in Fig. 2 restructuring Aminopeptidase A N_105 has to most substrate Catalytic activity, wherein catalytic activity highest substrate are L-Leu -4- nitroanilines (L-Leu-PNA), are secondly L- essence ammonia Acid -4- nitroanilines (L-Arg-PNA) and 1B -4- nitroanilines (L-Lys-PNA).

Embodiment 3 recombinates the measure of Aminopeptidase A N_105 optimal reactive temperatures

100mM Tris-HCl buffer solutions (pH7.0), using 1mM L-Leu -4- nitroanilines as substrate, add purifying The μ l of enzyme liquid 10 afterwards, determine light absorption value A at 20,25,30,35,40,45,50,55 and 60 DEG C respectively_405nm.As a result as shown in figure 3, Restructuring Aminopeptidase A N_105 still has higher catalytic activity at 25~50 DEG C, and optimal reactive temperature is 35 DEG C.

Embodiment 4 recombinates the measure of Aminopeptidase A N_105 optimal reaction pH values

Using 1mM L-Leu -4- nitroanilines as substrate, the μ l of enzyme liquid 10 after purification are added, respectively in pH4.0~7.0 Under the different pH condition such as (100mM phosphate buffers) and pH7.0~9.0 (Tris-HCl buffer solutions), 35 DEG C of measure extinctions Value A_405nm.As a result as shown in figure 4, restructuring Aminopeptidase A N_105 optimal reactions pH is 7.0, still there is higher enzyme in pH4.0~9.0 Vigor.

Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Sequence table

<110>Guangdong Industry Technical College

<120>A kind of aminopeptidase and its encoding gene and application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 495

<212> PRT

<213>Unknown (Unknown)

<400> 1

Met Asn Phe Gln Val Gln Asp Ala Ala Ile Val Ile Glu Ala Val Ile

1 5 10 15

Val Ala Leu Ala Phe Phe Ala Glu Lys Ile Ile Phe Ala Gln Gln Leu

20 25 30

Asp Ala Ala Ile Thr Gly Ser Leu Gln Ala Leu Leu Ala His Lys Gly

35 40 45

Ile Ser Thr Val Gly Tyr Ala Val Ser Leu Leu His Ser Leu Ala Gly

50 55 60

Leu Asn Val Asp Arg Tyr Tyr Val Gly Leu Glu Gly Gln Gln Leu Ala

65 70 75 80

Tyr Gly Ile Gln Thr Leu Leu Arg Lys Ala Gly Asp Ala Phe Thr Thr

85 90 95

Ile Lys Gly Gly Ala Val Ser Asp Glu Gln Ile Lys Leu Pro Ser Phe

100 105 110

Thr Lys Gln Arg Leu Asp Ala Ala Val Leu His Ile Ala Val Val Ser

115 120 125

Pro Gly His Gly Ala Tyr Glu Leu Thr Tyr Ser Lys Lys Lys Asp Lys

130 135 140

Gln Glu Leu Phe Glu Phe Thr Ala Ile Ala Leu Asp Asp Ala Gln Leu

145 150 155 160

Asp Leu Glu Gly Phe Ile Ser Val Gly Asp Val Thr Gly Val Leu Leu

165 170 175

Asn Phe Ala Arg Gln Leu Gly Asn Ala His Pro His Val Leu Thr Pro

180 185 190

Ala Ile Lys Thr Ala Glu Glu Ala Val Ala Leu Phe Gly Lys Thr Asp

195 200 205

Tyr Asp Val Lys Leu Asp Lys Glu Gln Met Glu Gly Ile Gly Asn Gly

210 215 220

Ser Leu Leu Ala Val Lys Lys Gly Ser Glu Asn Ile Pro Arg Leu Ile

225 230 235 240

Val Leu Thr Tyr Asn Gly Lys Glu Asp Ala Ala Pro Val Val Ala Leu

245 250 255

Val Gly Pro Gly Ile Thr Phe Asp Ser Gly Gly Tyr Ser Leu Lys Thr

260 265 270

Arg Gln Gly Met Glu Phe Met Lys Phe Asp Met Gly Cys Gly Ala Ala

275 280 285

Val Gly Gly Thr Val Glu Ala Ile Gly His Leu Leu Pro Pro Lys Asn

290 295 300

Ile Val Gly Val Ile Pro Ala Ser Asp Asn Ile Val Asn Gly Glu Ala

305 310 315 320

Ile Ser Pro Leu Asp Val Gly Thr Ser Leu Ser Gly Tyr Thr Ile Glu

325 330 335

Val Met Asn Thr Phe Pro Glu Gly Arg Leu Leu Leu Ala Asp Ala Val

340 345 350

Phe Tyr Ala Tyr Gln Asp Gly Pro Gly Val Leu Ala Asp Val Ala Thr

355 360 365

Leu Thr Gly Ala Tyr Ile Val Val Leu Gly Ala His Lys Thr Ala Gly

370 375 380

Ala Met Ile Asn Pro Gly Ile Leu Phe Asn Gln Leu Leu Asp Ala Ser

385 390 395 400

Glu Glu Val Asp Glu Pro Ala Gly Leu Pro Ala Ile Trp Gly Thr Glu

405 410 415

Gly Gly Val Val Thr Ser Asp Val Ala Asp Leu Pro Asn Tyr Gly Gly

420 425 430

Arg Asp Gly Thr Ala Leu Phe Glu Gly Met Phe Leu Thr Glu Glu Pro

435 440 445

Glu Asn Thr Pro Trp Leu Tyr Leu Asp Ile Ala Gly Gly Ser Arg Ile

450 455 460

Glu Asp Ala Val Leu Ile Gly Pro Asp Gly Ala Glu Gly Leu Leu Val

465 470 475 480

Pro Thr Leu Val Asp Leu Leu Thr Arg Glu Gly Glu Ala Glu Ala

485 490 495

<210> 2

<211> 1485

<212> DNA

<213>Unknown (Unknown)

<400> 2

atgaacttcc aggttcagga cgctgctatc gttatcgaag ctgttatcgt tgctctggct 60

ttcttcgctg aaaaaatcat cttcgctcag cagctggacg ctgctatcac cggttctctg 120

caggctctgc tggctcacaa aggtatctct accgttggtt acgctgtttc tctgctgcac 180

tctctggctg gtctgaacgt tgaccgttac tacgttggtc tggaaggtca gcagctggct 240

tacggtatcc agaccctgct gcgtaaagct ggtgacgctt tcaccaccat caaaggtggt 300

gctgtttctg acgaacagat caaactgccg tctttcacca aacagcgtct ggacgctgct 360

gttctgcaca tcgctgttgt ttctccgggt cacggtgctt acgaactgac ctactctaaa 420

aaaaaagaca aacaggaact gttcgaattc accgctatcg ctctggacga cgctcagctg 480

gacctggaag gtttcatctc tgttggtgac gttaccggtg ttctgctgaa cttcgctcgt 540

cagctgggta acgctcaccc gcacgttctg accccggcta tcaaaaccgc tgaagaagct 600

gttgctctgt tcggtaaaac cgactacgac gttaaactgg acaaagaaca gatggaaggt 660

atcggtaacg gttctctgct ggctgttaaa aaaggttctg aaaacatccc gcgtctgatc 720

gttctgacct acaacggtaa agaagacgct gctccggttg ttgctctggt tggtccgggt 780

atcaccttcg actctggtgg ttactctctg aaaacccgtc agggtatgga attcatgaaa 840

ttcgacatgg gttgcggtgc tgctgttggt ggtaccgttg aagctatcgg tcacctgctg 900

ccgccgaaaa acatcgttgg tgttatcccg gcttctgaca acatcgttaa cggtgaagct 960

atctctccgc tggacgttgg tacctctctg tctggttaca ccatcgaagt tatgaacacc 1020

ttcccggaag gtcgtctgct gctggctgac gctgttttct acgcttacca ggacggtccg 1080

ggtgttctgg ctgacgttgc taccctgacc ggtgcttaca tcgttgttct gggtgctcac 1140

aaaaccgctg gtgctatgat caacccgggt atcctgttca accagctgct ggacgcttct 1200

gaagaagttg acgaaccggc tggtctgccg gctatctggg gtaccgaagg tggtgttgtt 1260

acctctgacg ttgctgacct gccgaactac ggtggtcgtg acggtaccgc tctgttcgaa 1320

ggtatgttcc tgaccgaaga accggaaaac accccgtggc tgtacctgga catcgctggt 1380

ggttctcgta tcgaagacgc tgttctgatc ggtccggacg gtgctgaagg tctgctggtt 1440

ccgaccctgg ttgacctgct gacccgtgaa ggtgaagctg aagct 1485

<210> 3

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

cgcggatcca tgaacttcca ggttcag 27

<210> 4

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

cggaagctta gcttcacctt cacggtca 28

Claims

A kind of 1. aminopeptidase, it is characterised in that：Its amino acid sequence is as shown in SEQ ID NO.1.
A kind of 2. nucleotide sequence, it is characterised in that：It is the nucleotide sequence for encoding the aminopeptidase described in claim 1.
3. nucleotide sequence according to claim 2, it is characterised in that：Described nucleotide sequence be for intronless, Sequence with the complete opening code-reading frames of 1485bp.
4. nucleotide sequence according to claim 3, it is characterised in that：Described nucleotide sequence such as SEQ ID NO.2 It is shown.
5. the preparation method of the nucleotide sequence described in any one of claim 2~4, it is characterised in that comprise the following steps：

First, traditional fermented food environmental sample is extracted, total genome is extracted and purifies；

2nd, total genome after purification is handled through Sau3AI digestions；

3rd, digestion products are connected on pUC18/BamHI (BAP) carrier, tradition is built in electroporated bacillus coli DH 5 alpha Fermented food environment macro genomic library, aminopeptidase sun is screened from library with liquid selective medium and solid selection medium Property clone, positive clone molecule is obtained by high flux screening；

4th, compare design primer through sequencing and Blast, using the plasmid of positive clone molecule as template, carried out with designed primer Chain type enzymatic polymerization reacts, so as to clone to obtain described nucleotide sequence.
6. the preparation method of nucleotide sequence according to claim 5, it is characterised in that：

Traditional fermented food described in step 1 is one or more of soy sauce, fermented bean curd, beans sauce and sausage；

The composition of Selective agar medium described in step 3 is as follows：100 μ g/ml ampicillins, 10mmol/L L-Leus- Without amino acid yeast nitrogen, solvent is water by 4- nitroanilines, 1 (w/v) % glucose and 0.5 (w/v) %；

Designed primer described in step 4 is as follows：

Sense primer F1：5’-CGCGGATCCATGAACTTCCAGGTTCAG-3’；

Anti-sense primer F2：5’-CGGAAGCTTAGCTTCACCTTCACGGTCA-3’.
7. the preparation method of the aminopeptidase described in claim 1, it is characterised in that comprise the following steps：

(1) nucleotide sequence described in any one of claim 2~4 is cloned into expression vector, then is transferred in expression cell, Obtain the cell containing recombinant vector；

(2) cell containing recombinant vector obtained to step (1) is cultivated, and is induced through IPTG, is separated from culture, be pure Change, obtain aminopeptidase.
8. the preparation method of aminopeptidase according to claim 7, it is characterised in that：

Expression vector described in step (1) is pET-32a (+)；

Expression cell described in step (1) is e. coli bl21 (DE3)；

The culture medium of culture described in step (2) is the LB fluid nutrient mediums containing 100 μ g/ml ampicillins；

Induction described in step (2) comprises the following steps that：When the cell growth containing recombinant vector to OD₆₀₀Add when=1.0 Enter IPTG to final concentration 0.8mM, 20 DEG C, cultivate 20h under 200r/min.
9. application of the aminopeptidase described in claim 1 in flavor peptide is prepared.
10. application of the aminopeptidase according to claim 9 in flavor peptide is prepared, it is characterised in that comprise the following steps： Described aminopeptidase is added in the hydrolysis reaction system containing protein substrate, it is anti-that enzyme hydrolysis is carried out under the conditions of 25~35 DEG C 2~6h is answered, obtains corresponding flavor peptide.